Formin3 is required for assembly of the F-actin structure that mediates tracheal fusion in Drosophila

During tracheal development in Drosophila, some branches join to form a continuous luminal network. Specialized cells at the branch tip, called fusion cells, extend filopodia to make contact and become doughnut shaped to allow passage of the lumen. These morphogenetic processes accompany the highly regulated cytoskeletal reorganization of fusion cells. We identified the Drosophila formin3 (form3) gene that encodes a novel formin and plays a role in tracheal fusion. Formins are a family of proteins characterized by highly conserved formin homology (FH) domains. The formin family functions in various actin-based processes, including cytokinesis and cell polarity. During embryogenesis, form3 mRNA is expressed mainly in the tracheal system. In form3 mutant embryos, the tracheal fusion does not occur at some points. This phenotype is rescued by the forced expression of form3 in the trachea. We used live imaging of GFP-moesin during tracheal fusion to show that an F-actin structure that spans the adjoining fusion cells and mediates the luminal connection does not form at abnormal anastomosis sites in form3 mutants. These results suggested that Form3 plays a role in the F-actin assembly, which is essential for cellular rearrangement during tracheal fusion.     

Formin-1 protein associates with microtubules through a peptide domain encoded by exon-2

Formin family proteins coordinate actin filaments and microtubules. The mechanisms by which formins bind and regulate the actin cytoskeleton have recently been well defined. However, the molecular mechanism by which formins coordinate actin filaments and microtubules remains poorly understood. We demonstrate here that Isoform-Ib of the Formin-1 protein (Fmn1-Ib) binds to microtubules via a protein domain that is physically separated from the known actin-binding domains. When expressed at low levels in NIH3T3 fibroblasts, Fmn1-Ib protein localizes to cytoplasmic filaments that nocodazole disruption confirmed as interphase microtubules. A series of progressive mutants of Fmn1-Ib demonstrated that deletion of exon-2 caused dissociation from microtubules and a stronger association with actin membrane ruffles. The exon-2-encoded peptide binds purified tubulin in vitro and is also sufficient to localize GFP to microtubules. Exon-2 does not contain any known formin homology domains. Deletion of exon 5, 7, 8, the FH1 domain or FH2 domain did not affect microtubule binding. Thus, our results indicate that exon-2 of Fmn1-Ib encodes a novel microtubule-binding peptide. Since formin proteins associate with actin filaments through the FH1 and FH2 domains, binding to interphase microtubules through this exon-2-encoded domain provides a novel mechanism by which Fmn1-Ib could coordinate actin filaments and microtubules.     

芽殖酵母ASH1 mRNA的定位机制

mRNA定位是一种基因转录后水平的重要调控机制,对细胞的生理活动和分化发育都有着极其重要的作用。在芽殖酵母有丝分裂中,ASH1 mRNA在子细胞芽尖因不对称定位表达抑制了子细胞交配类型的转换。本综述介绍了芽殖酵母ASH1 mRNA定位的分子机制。     

Binary function of mRNA

Since the discovery of messenger RNA (mRNA) over half a century ago, the assumption has always been that the only function of mRNA is to make a protein. However, recent studies of prokaryotic and eukaryotic organisms unexpectedly show that some mRNAs may be functionally binary and have additional structural functions that are unrelated to their translation product. These findings imply that some of the phenotypic features of cells and organisms can also be binary, that is, they depend both on the function of a protein and the independent structural function of its mRNA. In this review, we will discuss this concept within the framework of multifunctional RNA molecules and the RNA World Hypothesis.     

Mutations to the Formin Homology 2 Domain of INF2 Protein Have Unexpected Effects on Actin Polymerization and Severing

INF2 (inverted formin 2) is a formin protein with unusual biochemical characteristics. As with other formins, the formin homology 2 (FH2) domain of INF2 accelerates actin filament assembly and remains at the barbed end, modulating elongation. The unique feature of INF2 is its ability to sever filaments and enhance depolymerization, which requires the C-terminal region. Physiologically, INF2 acts in the secretory pathway and is mutated in two human diseases, focal and segmental glomerulosclerosis and Charcot-Marie-Tooth disease. In this study, we investigate the effects of mutating two FH2 residues found to be key in other formins: Ile-643 and Lys-792. Surprisingly, neither mutation abolishes barbed end binding, as judged by pyrene-actin and total internal reflection (TIRF) microscopy elongation assays. The I643A mutation causes tight capping of a subset of filaments, whereas K792A causes slow elongation of all filaments. The I643A mutation has a minor inhibitory effect on polymerization activity but causes almost complete abolition of severing and depolymerization activity. The K792A mutation has relatively small effects on polymerization, severing, and depolymerization. In cells, the K792A mutant causes actin accumulation around the endoplasmic reticulum to a similar extent as wild type, whereas the I643A mutant causes no measurable polymerization. The inability of I643A to induce actin polymerization in cells is explained by its inability to promote robust actin polymerization in the presence of capping protein. These results highlight an important point: it is dangerous to assume that mutation of conserved FH2 residues will have equivalent effects in all formins. The work also suggests that both mutations have effects on the mechanism of processive elongation.     

Adaptations of cytoarchitecture in human dilated cardiomyopathy

Hypertrophic cardiomyopathy is characterised by a histological phenotype of myocyte disarray, but heart tissue samples from patients with dilated cardiomyopathy (DCM) often look comparatively similar to those from healthy individuals apart from conspicuous regions of fibrosis and necrosis. We have previously investigated subcellular alterations in the cytoarchitecture of mouse models of dilated cardiomyopathy and found that both the organisation and composition of the intercalated disc, i.e. the specialised type of cell–cell contact in the heart, is altered. There is also is a change in the composition of the M-band of the sarcomere due to an expression shift towards the more extensible embryonic heart (EH)-myomesin isoform. Analysis of human samples from the Sydney Human Heart Tissue Bank have revealed similar structural findings and also provided evidence for a dramatic change in overall cardiomyocyte size control, which has also been seen in the mouse. Together these changes in cytoarchitecture probably contribute to the decreased functional output that is seen in DCM.     

Ribonucleoprotein-dependent localization of the yeast class V myosin Myo4p

Class V myosins are motor proteins with functions in vesicle transport, organelle segregation, and RNA localization. Although they have been extensively studied, only little is known about the regulation of their spatial distribution. Here we demonstrate that a GFP fusion protein of the budding yeast class V myosin Myo4p accumulates at the bud cortex and is a component of highly dynamic cortical particles. Bud-specific enrichment depends on Myo4p's association with its cargo, a ribonucleoprotein complex containing the RNA-binding protein She2p. Cortical accumulation of Myo4p at the bud tip can be explained by a transient retention mechanism that requires SHE2 and, apparently, localized mRNAs bound to She2p. A mutant She2 protein that is unable to recognize its cognate target mRNA, ASH1, fails to localize Myo4p. Mutant She2p accumulates inside the nucleus, indicating that She2p shuttles between the nucleus and cytoplasm and is exported in an RNA-dependent manner. Consistently, inhibition of nuclear mRNA export results in nuclear accumulation of She2p and cytoplasmic Myo4p mislocalization. Loss of She2p can be complemented by direct targeting of a heterologous lacZ mRNA to a complex of Myo4p and its associated adaptor She3p, suggesting that She2p's function in Myo4p targeting is to link an mRNA to the motor complex.     

Determinants of Formin Homology 1 (FH1) domain function in actin filament elongation by formins

Formin-mediated elongation of actin filaments proceeds via association of Formin Homology 2 (FH2) domain dimers with the barbed end of the filament, allowing subunit addition while remaining processively attached to the end. The flexible Formin Homology 1 (FH1) domain, located directly N-terminal to the FH2 domain, contains one or more stretches of polyproline that bind the actin-binding protein profilin. Diffusion of FH1 domains brings associated profilin-actin complexes into contact with the FH2-bound barbed end of the filament, thereby enabling direct transfer of actin. We investigated how the organization of the FH1 domain of budding yeast formin Bni1p determines the rates of profilin-actin transfer onto the end of the filament. Each FH1 domain transfers actin to the barbed end independently of the other and structural evidence suggests a preference for actin delivery from each FH1 domain to the closest long-pitch helix of the filament. The transfer reaction is diffusion-limited and influenced by the affinities of the FH1 polyproline tracks for profilin. Position-specific sequence variations optimize the efficiency of FH1-stimulated polymerization by binding profilin weakly near the FH2 domain and binding profilin more strongly farther away. FH1 domains of many other formins follow this organizational trend. This particular sequence architecture may optimize the efficiency of FH1-stimulated elongation.     

An exclusively nuclear RNA-binding protein affects asymmetric localization of ASH1 mRNA and Ash1p in yeast

The localization of ASH1 mRNA to the distal tip of budding yeast cells is essential for the proper regulation of mating type switching in Saccharomyces cerevisiae. A localization element that is predominantly in the 3'-untranslated region (UTR) can direct this mRNA to the bud. Using this element in the three-hybrid in vivo RNA-binding assay, we identified a protein, Loc1p, that binds in vitro directly to the wild-type ASH1 3'-UTR RNA, but not to a mutant RNA incapable of localizing to the bud nor to several other mRNAs. LOC1 codes for a novel protein that recognizes double-stranded RNA structures and is required for efficient localization of ASH1 mRNA. Accordingly, Ash1p gets symmetrically distributed between daughter and mother cells in a loc1 strain. Surprisingly, Loc1p was found to be strictly nuclear, unlike other known RNA-binding proteins involved in mRNA localization which shuttle between the nucleus and the cytoplasm. We propose that efficient cytoplasmic ASH1 mRNA localization requires a previous interaction with specific nuclear factors.     

人类GABARAPL2基因的亚细胞定位

为了对GABARAPL2（GABAA受体相关蛋白相似蛋白2）基因的功能进行初步分析,首先通过同源比较的方法将序列与其同源物进行比较,发现GABARAPL2的氨基酸序列与GABARAP（GABAA受体相关蛋白）高度同源,而GABARAP已证实通过结合细胞骨架的微蛋白,使GABAA受体聚集,定位在细胞膜上,本文采用PCR法从人脑组织的cDNA文库中扩增出GABARAPL2的cDNA,克隆至T质粒载体中进行测序验证,然后以此为模板引物中引入酶切位点再次PCR,扩增出GABARAPL2的开放阅读框,并将其插入到加强型绿色荧光蛋白融合表达载体EGFP中,将绿色荧光蛋白标记的GABARAPL2和GABARAP分别转染HLF细胞株,结果两种蛋白的分布情况基本一致,在细胞质内和核内均有分布,而且核内的分布较胞质为多,结构功能域分析表明,GABARAPL2含有蛋白激酶C磷酸化位点和酪氨酸激酶磷酸化位点,可能通过磷酸化参与细胞骨架的变化,结论 GABARAPL2和GABARAP不仅在胞质中作为受体相关蛋白协助受体的聚集、定位,还参与体内许多其它重要的生理过程。     

FHOD1 formin is upregulated in melanomas and modifies proliferation and tumor growth

The functional properties of actin-regulating formin proteins are diverse and in many cases cell-type specific. FHOD1, a formin expressed predominantly in cells of mesenchymal lineage, bundles actin filaments and participates in maintenance of cell shape, migration and cellular protrusions. FHOD1 participates in cancer-associated epithelial to mesenchymal transition (EMT) in oral squamous cell carcinoma and breast cancer. The role of FHOD1 in melanomas has not been characterized. Here, we show that FHOD1 expression is typically strong in cutaneous melanomas and cultured melanoma cells while the expression is low or absent in benign nevi. By using shRNA to knockdown FHOD1 in melanoma cells, we discovered that FHOD1 depleted cells are larger, rounder and have smaller focal adhesions and inferior migratory capacity as compared to control cells. Importantly, we found FHOD1 depleted cells to have reduced colony-forming capacity and attenuated tumor growth in vivo, a finding best explained by the reduced proliferation rate caused by cell cycle arrest. Unexpectedly, FHOD1 depletion did not prevent invasive growth at the tumor margins. These results suggest that FHOD1 participates in key cellular processes that are dysregulated in malignancy, but may not be essential for melanoma cell invasion.     

The Role of Formin Tails in Actin Nucleation,Processive Elongation,and Filament Bundling

Formins are multidomain proteins that assemble actin in a wide variety of biological processes. They both nucleate and remain processively associated with growing filaments, in some cases accelerating filament growth. The well conserved formin homology 1 and 2 domains were originally thought to be solely responsible for these activities. Recently a role in nucleation was identified for the Diaphanous autoinhibitory domain (DAD), which is C-terminal to the formin homology 2 domain. The C-terminal tail of the Drosophila formin Cappuccino (Capu) is conserved among FMN formins but distinct from other formins. It does not have a DAD domain. Nevertheless, we find that Capu-tail plays a role in filament nucleation similar to that described for mDia1 and other formins. Building on this, replacement of Capu-tail with DADs from other formins tunes nucleation activity. Capu-tail has low-affinity interactions with both actin monomers and filaments. Removal of the tail reduces actin filament binding and bundling. Furthermore, when the tail is removed, we find that processivity is compromised. Despite decreased processivity, the elongation rate of filaments is unchanged. Again, replacement of Capu-tail with DADs from other formins tunes the processive association with the barbed end, indicating that this is a general role for formin tails. Our data show a role for the Capu-tail domain in assembling the actin cytoskeleton, largely mediated by electrostatic interactions. Because of its multifunctionality, the formin tail is a candidate for regulation by other proteins during cytoskeletal rearrangements.